Skeletal muscle weakness and fatigue is a common, debilitating condition in heart failure, sarcopenia, cachexia, muscular dystrophies, COPD, and other disorders. Na,K-ATPase activity is dramatically stimulated during muscle contraction and is absolutely required to maintain force and exercise performance. However, few studies have examined the role the Na,K-ATPase isoforms in muscle weakness and fatigue. Adult skeletal muscles express mainly the ?2 isoform of the Na,K-ATPase, in contrast to most other tissues which express mainly the ?1 isoform. The role of the ?2 isoform in skeletal muscle and the mechanisms by which its activity is stimulated are not known. The central hypotheses of this research is that the Na,K-ATPase ?2 isoform provides a reserve capacity which is largely inactive at rest but is rapidly stimulated during contraction, to meet the increased demand for Na/K transport; and that its regulation is achieved in part by phosphorylation of the muscle-specific FXYD1 subunit of the Na,K-ATPase. This hypothesis will be tested using a novel gene targeted mouse which specifically lacks the Na,K-ATPase ?2 in adult skeletal muscles (sk??2-/-) and shows marked exercise intolerance and skeletal muscle weakness. The Specific Aims are to: 1) Determine the acute role of the Na,K-ATPase ?2 enzyme in maintaining force and exercise performance; 2) Determine the role of the Na,K-ATPase ?2 enzyme in membrane excitation in the transverse tubules and resistance to fatigue; and 3) a, Define the role of FXYD1 phosphorylation in the acute stimulation of Na,K-ATPase ?2 activity by ?2- adrenergic receptor activation during contraction; b, Determine the contribution of altered Na,K-ATPase content or function to muscle fatigue and exercise intolerance in heart failure. These Aims will be approached using an integrated strategy which combines genetic manipulations in the mouse with functional measurements from the animal to the cellular and subcellular level. Collectively, this project will advance our understanding of the physiological roles of the Na,K-ATPase ?2 isoform and the mechanisms by which it is regulated in skeletal muscle, and identify new molecular targets for therapeutic interventions in muscle weakness and fatigue.